US4200435A - Determination of glycosylated hemoglobin in blood - Google Patents
Determination of glycosylated hemoglobin in blood Download PDFInfo
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- US4200435A US4200435A US05/973,368 US97336878A US4200435A US 4200435 A US4200435 A US 4200435A US 97336878 A US97336878 A US 97336878A US 4200435 A US4200435 A US 4200435A
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- hemoglobin
- allosteric
- hemoglobins
- glycosylated
- glycosylated hemoglobin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
- G01N33/721—Haemoglobin
- G01N33/723—Glycosylated haemoglobin
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- H—ELECTRICITY
- H04—ELECTRIC COMMUNICATION TECHNIQUE
- H04M—TELEPHONIC COMMUNICATION
- H04M19/00—Current supply arrangements for telephone systems
- H04M19/02—Current supply arrangements for telephone systems providing ringing current or supervisory tones, e.g. dialling tone or busy tone
- H04M19/04—Current supply arrangements for telephone systems providing ringing current or supervisory tones, e.g. dialling tone or busy tone the ringing-current being generated at the substations
Definitions
- Hemoglobin exists in two allosteric forms.
- the T (taught) and the R (relaxed) form have different chemical and physical properties and the relative amounts of R and T hemoglobin can be determined by art recognized techniques such as ultraviolet, infrared, visible, nuclear magnetic resonance, and electron spin resonance spectroscopy.
- absorption spectra of hemoglobin derivatives i.e., R ⁇ T transition as a function of ligand and inositol hexaphosphate binding.
- Circular dichroism and chemical reactivity are among other techniques for distinguishing R and T states of hemoglobin.
- the relative amount of R and T states can be determined by both end-point and kinetic techniques.
- Glycosylated hemoglobin is known to be associated with diabetes mellitus. Glycosylated hemoglobin is present in non-diabetics at a level of about 5% of total hemoglobin, while diabetics have 2-4 times that amount (Science, 200, Apr. 7, 1978). Glycosylated hemoglobin level provides an index of a patient's average blood glucose concentration over a long period of time. This index is not affected by short-term fluctuations in blood sugar (hour-to-hour) and, hence, gives a relatively precise reflection of the state of blood glucose control in diabetics.
- HbA Glycosylated hemoglobin
- the percent of glycosylated hemoglobin in blood can be measured by monitoring the shift in the equilibrium populations of R and T allosteric forms of hemoglobins when the non-glycosylated hemoglobin is reacted with an allosteric site binding substance. This reaction causes a shift from the R to the T allosteric form in the non-glycosylated fraction portion of the hemoglobin.
- the glycosylated hemoglobin in the blood sample does not contribute to the shaft in the equilibrium of the allosteric forms since glycosylation blocks the allosteric binding site.
- the higher the percentage of glycosylated hemoglobin in the blood sample the smaller the shift between allosteric forms upon reacting the hemoglobins with an allosteric site binding substance.
- the present invention takes advantage of the reactivity of the allosteric binding site which is accessible in non-glycosylated hemoglobin and the resulting shift in the equilibrium of allosteric forms of the glycosylate and non-glycosylated hemoglobin mixture resulting when an allosteric binding site substance is reacted with the non-glycosylated hemoglobin fraction.
- the present invention encompasses a method for determining glycosylated hemoglobin in blood samples which involves liberating hemoglobins from red blood cells by chemical or physical means and reacting non-glycosylated hemoglobin with an allosteric site binding substance which reacts with the allosteric binding site of non-glycosylated hemoglobin and thereby alters the distribution between allosteric forms of the hemoglobins and measuring the change.
- the present invention provides methods and reagents for a clinical assay of glycosylated hemoglobin in blood samples.
- organophosphates, sulfates, carboxylic acids represented by inositol hexaphosphate, J. Biol. Chem., 246, 7168 (1971); 2,3-diphosphoglycerate, Nature, 234, 174 (1971); adenosine triphosphate, Biochem. Biophys. Res. Comm., 26, 162 (1967); pyridoxal phosphate, Fed. Proc. Fed. Amer. Soc., Expl. Biol., 28, 604 (1969); inositol hexasulfate, Biochemistry, 15, 3396 (1976); inositol pentaphosphate, Can.
- Inositol hexaphosphate is a preferred allosteric effector site binding substance.
- red blood cells It is generally desirable to lyse red blood cells to release hemoglobins.
- Common cationic (e.g., cetyl tri-methyl ammonium bromide); anionic (e.g., sodium dodecylsulfate and sodium deoxycholate) and neutral (e.g., saponin and octyl phenoxypolyethoxyethanol) detergents are useful in lysing red blood cells.
- neutral detergents in the concentration range of about 0.025 to 0.5 volume percent are preferred.
- Mechanical rupture for example ultrasonication and hypotonic lysis, are also effective ways of releasing hemoglobin from red blood cells.
- Binding of heme-binding ligands to heme iron generally shifts the equilibrium of allosteric hemoglobin isomers to the relaxed (R) form.
- R relaxed
- heme-binding moiety of the hemoglobins in the test sample is coordinated with a heme-binding ligand larger shifts in the equilibrium populations of allosteric forms of hemoglobin are observed.
- This magnification in shift in equilibrium enhances accuracy and precision of glycosylated hemoglobin determination.
- This coordination of heme-binding ligand to shift equilibrium of allosteric isomers is applicable when the iron is in the Fe +2 or the Fe +3 (methemoglobin) states.
- hemoglobin Those skilled in the hemoglobin arts will recognize a wide variety of heme-binding ligands which bind to the iron of hemoglobin or methemoglobin.
- isocyanides such as alkyl isocyanides having 1-6 carbon atoms or phenyl isocyanides are particularly desirable heme-binding ligands for hemoglobin in the Fe +2 state.
- suitable ligands are O 2 and NO.
- oxyhemoglobin (glycosylated and non-glycosylated) is preferably deoxygenated by reaction with sodium dithionite or other well-known reducing agents to deoxyhemoglobin.
- the deoxyhemoglobin is reacted with alkylisocyanide such as n-butylisocyanide and as a result reaction with an allosteric effector site binding ligand provides a more definitive shift in equilibrium of the allosteric forms permitting determination of glycosylated hemoglobin.
- Hemoglobin is oxidized to methemoglobin by art recognized techniques, Antonini and Brunoni, Hemoglobin and Myoglobin in Their Reactions With Ligands, North Holland Publishing Co., Amsterdam (1971).
- potassium ferricyanide, sodium nitrite, aniline, and phenylhydrazine are convenient reagents for oxidizing hemoglobin to methemoglobin.
- Autooxidation in the presence of dyes such as methylene blue also oxidizes hemoglobin to methemoglobin.
- Non-glycosylated methemoglobin is reactive with allosteric effector site binding substances described for non-glycosylated hemoglobin.
- heme-binding ligands which bind with methemoglobin. These ligands include cyanate, thiocyanate, N-hydroxyacetamide, imidazole and derivatives thereof. Perutz et al., Biochemistry, 17, 3640-3652 (1978).
- ligands are fluoride, azide, nitrite, cyanide, water, hydroxide ammonia, acetate and formate.
- Imidazole at about 0.1 M is a preferred heme-binding ligand for use with methemoglobin.
- a reagent which is 0.1 M imidazole, 0.2 mM potassium ferricyanide, K 3 Fe(CN) 6 , and 0.05% by volume triton x-100 (octyl phenoxypolyethoxyethanol) detergent in buffer at pH 6.8 is added to 10-20 ⁇ l of whole blood and the mixture is incubated for ten minutes.
- a reagent which is 0.1 M imidazole, 0.2 mM potassium ferricyanide, K 3 Fe(CN) 6 , and 0.05% by volume triton x-100 (octyl phenoxypolyethoxyethanol) detergent in buffer at pH 6.8 is added to 10-20 ⁇ l of whole blood and the mixture is incubated for ten minutes.
- the potassium ferricyanide oxidizes the hemoglobin to methemoglobin; the triton x-100 is a neutral detergent which lyses the cells to release hemoglobins; and the imidazole coordinates with the iron shifting equilibrium allosteric isomers to the (R) form.
- the absorption spectrum of this mixture is recorded at 560 nm and 635 nm. Then 2 ⁇ l of a 0.1 M inositol hexaphosphate solution, pH 6.8 is added. The latter reagent reacts with the allosteric binding site is non-glycosylated hemoglobin and shifts equilibrium of the allosteric isomers to the (T) target form. The absorption specturm at 560 nm and 635 nm is measured again. Glycosylated hemoglobin concentration is reflected by a decrease in 560 nm absorption and increased in the 635 nm absorption.
- the present invention also includes test kits for determining glycosylated hemoglobin in blood samples.
- the test kit includes separate or in combination a red blood cell lysing agent, an oxidizing agent for oxidizing hemoglobin to methemoglobin, a heme-binding ligand, and an allosteric site binding substance.
- the test kit will generally contain controls or standards.
- the reagents may be separate, combined into two reagents as shown in Example 1, or a single reagent as illustrated in Example 2. Those skilled in the analytical arts will recognize that these reagents may be added individually or in combination in sequence or simultaneously.
- a preferred test kit consists of a reagent of 0.1 M imidazole, 0.2 mM potassium ferricyanide, and 0.05% by volume triton x-100, pH 6.8, and another reagent of 0.1 M inositol hexaphosphate, pH 6.8.
- This kit will generally contain standards having between 0-100% glycosylated hemoglobin as well as controls having a known amount of glycosylated hemoglobin; the controls being in the normal range and some in the abnormal range.
- the present invention further encompasses reagents comprising two or more of (a) a red blood cell lysing agent, (b) an oxidizing agent for oxidizing hemoglobin to methemoglobin, (c) a heme-binding ligand, and (d) an allosteric site binding substance in water or aqueous buffer as diluent, the pH being about 6 to 8, preferrably about 6.8.
- reagents comprising two or more of (a) a red blood cell lysing agent, (b) an oxidizing agent for oxidizing hemoglobin to methemoglobin, (c) a heme-binding ligand, and (d) an allosteric site binding substance in water or aqueous buffer as diluent, the pH being about 6 to 8, preferrably about 6.8.
- reagents comprising two or more of (a) a red blood cell lysing agent, (b) an oxidizing agent for oxidizing hemoglobin
- Reagent A 0.1 M imidazole, 0.2 mM K 3 Fe(CN) 6 , 0.05%v/v triton x-100 (octyl phenoxypolyethoxyethanol detergent), in water, pH 6.8
- Reagent B 0.1 M inositol hexaphosphate (IHP), in water, pH 6.8
- a single reagent addition is used by taking advantage of isosbestic points for the IHP effect to normalize for hemoglobin concentration.
- Reagent C To 1 vol. of Reagent B add 500 volumes Reagent A from Example 1
- Reagent A 50 mM bis-tris buffer [bis-(2-hydroxethyl)imino-tris-(hydroxymethyl)methane]; 0.05%v/v triton x-100; 1 mM n-butyl isocyanide; and 2 mg/ml sodium dithionite in water, pH 6.8
- Reagent B 2.5 mM inositol hexaphosphate (IHP), in water, pH 6.8
- a purified sample of hemoglobin A is mixed with various amounts of purified glycosylated hemoglobin to give hemoglobin samples containing known amounts of glycoslated hemoglobin.
- Reagent A further containing 0.05% (triton x-100 detergent) is added to 10-20 ⁇ l of whole-blood and the analysis is run as above to determine unknown glycosylated hemoglobin.
Abstract
A method for determining glycosylated hemoglobin in blood samples which involves liberating hemoglobins from red blood cells by chemical or physical means and reacting non-glycosylated hemoglobin with an allosteric site binding substance which reacts with the allosteric binding site of non-glycosylated hemoglobin and thereby alters the distribution between allosteric forms of the hemoglobins and measuring the change. This method is useful in monitoring glucose metabolism for detecting and controlling diabetes.
Description
Hemoglobin exists in two allosteric forms. The T (taught) and the R (relaxed) form. These forms have different chemical and physical properties and the relative amounts of R and T hemoglobin can be determined by art recognized techniques such as ultraviolet, infrared, visible, nuclear magnetic resonance, and electron spin resonance spectroscopy. For example, Perutz et al., Biochem., No. 17, 3641 (1978) describes absorption spectra of hemoglobin derivatives, i.e., R→T transition as a function of ligand and inositol hexaphosphate binding. Circular dichroism and chemical reactivity are among other techniques for distinguishing R and T states of hemoglobin. The relative amount of R and T states can be determined by both end-point and kinetic techniques.
Elevated levels of glycosylated hemoglobin are known to be associated with diabetes mellitus. Glycosylated hemoglobin is present in non-diabetics at a level of about 5% of total hemoglobin, while diabetics have 2-4 times that amount (Science, 200, Apr. 7, 1978). Glycosylated hemoglobin level provides an index of a patient's average blood glucose concentration over a long period of time. This index is not affected by short-term fluctuations in blood sugar (hour-to-hour) and, hence, gives a relatively precise reflection of the state of blood glucose control in diabetics.
Glycosylated hemoglobin is commonly referred to as HbA or fast hemoglobin because it migrates faster on a chromatograph column and, indeed, is generally measured by chromatography or electrophoresis.
It has been discovered that the percent of glycosylated hemoglobin in blood can be measured by monitoring the shift in the equilibrium populations of R and T allosteric forms of hemoglobins when the non-glycosylated hemoglobin is reacted with an allosteric site binding substance. This reaction causes a shift from the R to the T allosteric form in the non-glycosylated fraction portion of the hemoglobin. The glycosylated hemoglobin in the blood sample does not contribute to the shaft in the equilibrium of the allosteric forms since glycosylation blocks the allosteric binding site. Thus, the higher the percentage of glycosylated hemoglobin in the blood sample, the smaller the shift between allosteric forms upon reacting the hemoglobins with an allosteric site binding substance. The present invention takes advantage of the reactivity of the allosteric binding site which is accessible in non-glycosylated hemoglobin and the resulting shift in the equilibrium of allosteric forms of the glycosylate and non-glycosylated hemoglobin mixture resulting when an allosteric binding site substance is reacted with the non-glycosylated hemoglobin fraction.
The present invention encompasses a method for determining glycosylated hemoglobin in blood samples which involves liberating hemoglobins from red blood cells by chemical or physical means and reacting non-glycosylated hemoglobin with an allosteric site binding substance which reacts with the allosteric binding site of non-glycosylated hemoglobin and thereby alters the distribution between allosteric forms of the hemoglobins and measuring the change. Unexpectedly, the present invention provides methods and reagents for a clinical assay of glycosylated hemoglobin in blood samples.
A wide variety of compounds are known as effective allosteric effector site binding substances. These include organophosphates, sulfates, carboxylic acids represented by inositol hexaphosphate, J. Biol. Chem., 246, 7168 (1971); 2,3-diphosphoglycerate, Nature, 234, 174 (1971); adenosine triphosphate, Biochem. Biophys. Res. Comm., 26, 162 (1967); pyridoxal phosphate, Fed. Proc. Fed. Amer. Soc., Expl. Biol., 28, 604 (1969); inositol hexasulfate, Biochemistry, 15, 3396 (1976); inositol pentaphosphate, Can. J. Chem., 47, 63 (1969); 8-hydroxy-1,3,6-pyrenetrisulfonate, J. Biol. Chem., 246, 5832 (1971); O-iodosodium benzoate, The Journal of Pharmacology and Experimental Therapeutics, 203, 72 (1977). Those skilled in the hemoglobin arts will recognize a wide variety of effector site binding substances equivalent for practicing the present invention. Inositol hexaphosphate is a preferred allosteric effector site binding substance.
It is generally desirable to lyse red blood cells to release hemoglobins. Common cationic (e.g., cetyl tri-methyl ammonium bromide); anionic (e.g., sodium dodecylsulfate and sodium deoxycholate) and neutral (e.g., saponin and octyl phenoxypolyethoxyethanol) detergents are useful in lysing red blood cells. Neutral detergents in the concentration range of about 0.025 to 0.5 volume percent are preferred. Mechanical rupture, for example ultrasonication and hypotonic lysis, are also effective ways of releasing hemoglobin from red blood cells.
Binding of heme-binding ligands to heme iron generally shifts the equilibrium of allosteric hemoglobin isomers to the relaxed (R) form. Thus, when the heme-binding moiety of the hemoglobins in the test sample is coordinated with a heme-binding ligand larger shifts in the equilibrium populations of allosteric forms of hemoglobin are observed. This magnification in shift in equilibrium enhances accuracy and precision of glycosylated hemoglobin determination. This coordination of heme-binding ligand to shift equilibrium of allosteric isomers is applicable when the iron is in the Fe+2 or the Fe+3 (methemoglobin) states.
Those skilled in the hemoglobin arts will recognize a wide variety of heme-binding ligands which bind to the iron of hemoglobin or methemoglobin.
For example, isocyanides such as alkyl isocyanides having 1-6 carbon atoms or phenyl isocyanides are particularly desirable heme-binding ligands for hemoglobin in the Fe+2 state. Other suitable ligands are O2 and NO.
It is generally preferred to have a single ligand bound to iron since this results in simpler measurements of the shift in allosteric forms. For example, oxyhemoglobin (glycosylated and non-glycosylated) is preferably deoxygenated by reaction with sodium dithionite or other well-known reducing agents to deoxyhemoglobin. The deoxyhemoglobin is reacted with alkylisocyanide such as n-butylisocyanide and as a result reaction with an allosteric effector site binding ligand provides a more definitive shift in equilibrium of the allosteric forms permitting determination of glycosylated hemoglobin.
Hemoglobin is oxidized to methemoglobin by art recognized techniques, Antonini and Brunoni, Hemoglobin and Myoglobin in Their Reactions With Ligands, North Holland Publishing Co., Amsterdam (1971). Thus, potassium ferricyanide, sodium nitrite, aniline, and phenylhydrazine are convenient reagents for oxidizing hemoglobin to methemoglobin. Autooxidation in the presence of dyes such as methylene blue also oxidizes hemoglobin to methemoglobin.
Non-glycosylated methemoglobin is reactive with allosteric effector site binding substances described for non-glycosylated hemoglobin.
Those skilled in the hemoglobin arts will recognize a large variety of heme-binding ligands which bind with methemoglobin. These ligands include cyanate, thiocyanate, N-hydroxyacetamide, imidazole and derivatives thereof. Perutz et al., Biochemistry, 17, 3640-3652 (1978).
Other common ligands are fluoride, azide, nitrite, cyanide, water, hydroxide ammonia, acetate and formate. Imidazole at about 0.1 M is a preferred heme-binding ligand for use with methemoglobin.
In a preferred embodiment, 1 ml of a reagent which is 0.1 M imidazole, 0.2 mM potassium ferricyanide, K3 Fe(CN)6, and 0.05% by volume triton x-100 (octyl phenoxypolyethoxyethanol) detergent in buffer at pH 6.8 is added to 10-20 μl of whole blood and the mixture is incubated for ten minutes.
The potassium ferricyanide oxidizes the hemoglobin to methemoglobin; the triton x-100 is a neutral detergent which lyses the cells to release hemoglobins; and the imidazole coordinates with the iron shifting equilibrium allosteric isomers to the (R) form.
The absorption spectrum of this mixture is recorded at 560 nm and 635 nm. Then 2 μl of a 0.1 M inositol hexaphosphate solution, pH 6.8 is added. The latter reagent reacts with the allosteric binding site is non-glycosylated hemoglobin and shifts equilibrium of the allosteric isomers to the (T) target form. The absorption specturm at 560 nm and 635 nm is measured again. Glycosylated hemoglobin concentration is reflected by a decrease in 560 nm absorption and increased in the 635 nm absorption.
The present invention also includes test kits for determining glycosylated hemoglobin in blood samples. The test kit includes separate or in combination a red blood cell lysing agent, an oxidizing agent for oxidizing hemoglobin to methemoglobin, a heme-binding ligand, and an allosteric site binding substance. The test kit will generally contain controls or standards. The reagents may be separate, combined into two reagents as shown in Example 1, or a single reagent as illustrated in Example 2. Those skilled in the analytical arts will recognize that these reagents may be added individually or in combination in sequence or simultaneously. A preferred test kit consists of a reagent of 0.1 M imidazole, 0.2 mM potassium ferricyanide, and 0.05% by volume triton x-100, pH 6.8, and another reagent of 0.1 M inositol hexaphosphate, pH 6.8. This kit will generally contain standards having between 0-100% glycosylated hemoglobin as well as controls having a known amount of glycosylated hemoglobin; the controls being in the normal range and some in the abnormal range.
The present invention further encompasses reagents comprising two or more of (a) a red blood cell lysing agent, (b) an oxidizing agent for oxidizing hemoglobin to methemoglobin, (c) a heme-binding ligand, and (d) an allosteric site binding substance in water or aqueous buffer as diluent, the pH being about 6 to 8, preferrably about 6.8. The combinations of (a)+(b); (a)+(b)+(c); and (a)+(b)+(c)+(d) in in diluent are preferred reagents.
The hereinafter set out examples are intended to illustrate the present invention and not limit it in spirit or scope.
Reagent A: 0.1 M imidazole, 0.2 mM K3 Fe(CN)6, 0.05%v/v triton x-100 (octyl phenoxypolyethoxyethanol detergent), in water, pH 6.8
Reagent B: 0.1 M inositol hexaphosphate (IHP), in water, pH 6.8
To 1.0 ml of Reagent A at 25° C. add 10-20 μl whole blood, incubate 10 minutes to allow for cell lysis and oxidation of hemoglobin to methemoglobin. Record visible spectrum, 450 nm to 700 nm, specifically monitoring absorbance at 560 nm and 635 nm. Then add 2 μl Reagent B to the reaction mixture. Record another spectrum as before.
Standards are prepared by spiking whole blood with glycosylated hemoglobin.
______________________________________
RESULTS
Standard Curve
Normalized
Difference - % Glycosylated Hb No
IHP A.sup. 560nm A.sup.635nm + IHP
A.sup.560nm A.sup. 635nm
##STR1##
______________________________________
0% 0.664 0.089 0.592 0.123 0.184
5% 0.654 0.086 0.588 0.120 0.176
10% 0.657 0.089 0.593 0.121 0.169
15% 0.658 0.090 0.596 0.118 0.158
20% 0.663 0.095 0.609 0.123 0.144
25% 0.651 0.091 0.600 0.117 0.138
50% 0.645 0.098 0.611 0.113 0.090
100% 0.717 0.123 0.715 0.128 0.012
______________________________________
Calculations: Δ = A.sup.560nm - A.sup.635nm
##STR2##
______________________________________
No IHP A.sup.560nm A.sup. 635nm
+ IHP A.sup.560nm A.sup.635nm
##STR3##
______________________________________
Unknown 0.705 0.098 0.637 0.135 0.173
(whole blood)
##STR4##
glycosylated
Hb
______________________________________
Check from column method (commercially available from Helena
and ISOLAB)
##STR5##
______________________________________
A single reagent addition is used by taking advantage of isosbestic points for the IHP effect to normalize for hemoglobin concentration.
Reagent C: To 1 vol. of Reagent B add 500 volumes Reagent A from Example 1
To 1.0 mlReagent C add 10-20 μl whole blood. Incubate 10 minutes to allow for lysis, oxidation of hemoglobin to methemoglobin, and reaction of methemoglobin with imidazole and IHP. Record visible spectrum 450 nm to 700 nm, especially monitoring 476 nm, 560 nm, 635 nm, and 700 nm.
476 nm and 700 nm are isosbestic wavelength for the IHP effect.
Calculation: Normalized ΔΔ=A560 -A635 /A476 -A700
______________________________________
RESULTS
Standard Curve
% Glycosylated Normalized
Hemoglobin A.sup.476
A.sup.560
A.sup.635
A.sup.700
ΔΔ
______________________________________
0% .608 .592 .123 .016 .792
5% .598 .588 .120 .018 .807
10% .602 .593 .121 .020 .811
15% .598 .596 .118 .019 .826
20% .613 .609 .123 .024 .825
25% .603 .600 .117 .022 .831
50% .598 .611 .113 .026 .871
100% .685 .715 .128 .044 .916
Typical Normal
Unknown .653 .637 .135 .030 .806
5%
______________________________________
Reagent A: 50 mM bis-tris buffer [bis-(2-hydroxethyl)imino-tris-(hydroxymethyl)methane]; 0.05%v/v triton x-100; 1 mM n-butyl isocyanide; and 2 mg/ml sodium dithionite in water, pH 6.8
Reagent B: 2.5 mM inositol hexaphosphate (IHP), in water, pH 6.8
A purified sample of hemoglobin A is mixed with various amounts of purified glycosylated hemoglobin to give hemoglobin samples containing known amounts of glycoslated hemoglobin.
100 μl of various hemoglobin samples are placed in a cuvette and 1.0 ml of Reagent A was added. The absorbance at 530 nm and 585 nm is read after an incubation of about 2 minutes.
After the initial readings at 530 nm and 585 nm, 10 μl of Reagent B is added and after an incubation of about 1 min. the absorbance at 530 nm and 585 nm is again read.
______________________________________
RESULTS
% Glycosylated Hemoglobin
No IHP A.sup.530 A.sup.585
+ IHP A.sup.530 A.sup.585
##STR6##
______________________________________
0% .529 .153 .369 .261 .287
5% .511 .159 .363 .261 .290
10% .489 .174 .368 .260 .343
15% .478 .177 .362 .258 .346
20% .487 .191 .377 .268 .368
25% .460 .191 .361 .258 .383
______________________________________
Reagent A further containing 0.05% (triton x-100 detergent) is added to 10-20 μl of whole-blood and the analysis is run as above to determine unknown glycosylated hemoglobin.
Claims (6)
1. A method for determining glycosylated hemoglobin in a blood sample comprising chemically or physically treating the blood sample to release glycosylated and non-glycosylated hemoglobins from red blood cells, intermixing an allosteric effector site binding substance which binds to the allosteric effector site of the non-glycosylated hemoglobin thereby changing the distribution between allosteric forms of the hemoglobins, and measuring the change.
2. The method according to claim 1 wherein the heme moiety of the hemoglobin is coordinated with a hemebinding ligand.
3. The method according to claim 1 wherein the hemoglobins are oxidized to methemoglobin.
4. The method according to claim 1 wherein the blood sample is lysed with a detergent to release hemoglobins from blood cells.
5. The method according to claim 1 wherein the change in distribution of the allosteric forms of hemoglobin is measured by absorption spectroscopy in the range of 450 nm-700 nm.
6. A method for determining glycosylated hemoglobin in a blood sample comprising:
(a) lysing red blood cells to release glycosylated and nonglycosylated hemoglobins;
(b) oxidizing the hemoglobins to methemoglobins and coordinating the heme moiety with a heme-binding ligand;
(c) intermixing an allosteric effector site binding substance which binds to the allosteric effector site of the nonglycosylated hemoglobin thereby changing the distribution between allosteric forms of the hemoglobins; and
(d) measuring the change by adsorption spectroscopy in the range of 450 nm-700 nm.
Priority Applications (15)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US05/973,368 US4200435A (en) | 1978-12-26 | 1978-12-26 | Determination of glycosylated hemoglobin in blood |
| CA000338610A CA1119081A (en) | 1978-12-26 | 1979-10-29 | Determination of glycosylated hemoglobin in blood |
| ZA00795808A ZA795808B (en) | 1978-12-26 | 1979-10-30 | Determination of glycosylated hemoglobin in blood |
| GB7937929A GB2038476B (en) | 1978-12-26 | 1979-11-01 | Determination of glycosylated hemoglobin in blood |
| NL7908272A NL7908272A (en) | 1978-12-26 | 1979-11-12 | DETERMINATION OF GLYCOSYLATED HEMOGLOBINE IN BLOOD. |
| AU52754/79A AU528008B2 (en) | 1978-12-26 | 1979-11-13 | Determination of glycosylated haemoglobin |
| DE2950457A DE2950457B2 (en) | 1978-12-26 | 1979-12-14 | Method for the determination of glycosylated hemoglobin in blood and reagent for carrying out the method |
| SE7910340A SE445147B (en) | 1978-12-26 | 1979-12-14 | PROCEDURE FOR DETERMINING GLYCOSYLATED HEMOGLOBIN IN A BLOOD SAMPLE AND TEST PACKAGING TO PERFORM THE PROCEDURE |
| JP16581279A JPS5587954A (en) | 1978-12-26 | 1979-12-21 | Measuring glucocyl hemoglobin in blood |
| IT28370/79A IT1198318B (en) | 1978-12-26 | 1979-12-21 | PROCEDURE FOR DETERMINING GLYCOSYLATED HEMOGLOBIN IN BLOOD SAMPLES |
| CH1142879A CH641571A5 (en) | 1978-12-26 | 1979-12-21 | METHOD AND REAGENTS FOR THE DETERMINATION OF GLUCOSYLATED HEMOGLOBIN IN BLOOD. |
| AT808579A AT362534B (en) | 1978-12-26 | 1979-12-21 | METHOD FOR PRODUCING GLYCOSYLATED HAEMOGLOBIN IN BLOOD AND REAGENTS FOR CARRYING OUT THE METHOD |
| BE0/198711A BE880817A (en) | 1978-12-26 | 1979-12-21 | REAGENT PROCESS FOR THE DETERMINATION OF GLUCOSYLATED HEMOGLOBIN IN BLOOD |
| FR7931601A FR2445527A1 (en) | 1978-12-26 | 1979-12-24 | METHOD AND REAGENTS FOR THE DETERMINATION OF GLUCOSYLATED HEMOGLOBIN IN BLOOD |
| ES487265A ES8103381A1 (en) | 1978-12-26 | 1979-12-26 | Determination of glycosylated hemoglobin in blood |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US05/973,368 US4200435A (en) | 1978-12-26 | 1978-12-26 | Determination of glycosylated hemoglobin in blood |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| US06/087,367 Continuation US4255385A (en) | 1979-10-23 | 1979-10-23 | Reagent and test kit for determining glycosylated hemoglobin |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| US4200435A true US4200435A (en) | 1980-04-29 |
Family
ID=25520823
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| US05/973,368 Expired - Lifetime US4200435A (en) | 1978-12-26 | 1978-12-26 | Determination of glycosylated hemoglobin in blood |
Country Status (15)
| Country | Link |
|---|---|
| US (1) | US4200435A (en) |
| JP (1) | JPS5587954A (en) |
| AT (1) | AT362534B (en) |
| AU (1) | AU528008B2 (en) |
| BE (1) | BE880817A (en) |
| CA (1) | CA1119081A (en) |
| CH (1) | CH641571A5 (en) |
| DE (1) | DE2950457B2 (en) |
| ES (1) | ES8103381A1 (en) |
| FR (1) | FR2445527A1 (en) |
| GB (1) | GB2038476B (en) |
| IT (1) | IT1198318B (en) |
| NL (1) | NL7908272A (en) |
| SE (1) | SE445147B (en) |
| ZA (1) | ZA795808B (en) |
Cited By (20)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1981000913A1 (en) * | 1979-09-24 | 1981-04-02 | J Graas | Microchromatographic device and method for rapid determination of a desired substance |
| US4260516A (en) * | 1979-12-07 | 1981-04-07 | Abbott Laboratories | Glycosylated hemoglobin standards |
| US4274978A (en) * | 1979-12-07 | 1981-06-23 | Abbott Laboratories | Standards for determining glycosylated hemoglobin |
| US4297238A (en) * | 1978-11-22 | 1981-10-27 | Merck Patent Gesellschaft Mit Beschrankter Haftung | Hemolysing solution preparing hemolysed blood having a stabilized glucose content |
| EP0072440A1 (en) * | 1981-07-16 | 1983-02-23 | Sherwood Medical Industries Inc. | Standard or control material for glycosylated or total hemoglobin determination |
| US4438204A (en) | 1981-10-16 | 1984-03-20 | Boehringer Mannheim Gmbh | Determination of glycosilated hemoglobin |
| US4629692A (en) * | 1982-12-06 | 1986-12-16 | Miles Laboratories, Inc. | Immunoassay for nonenzymatically glucosylated proteins and protein fragments an index of glycemia |
| US4642295A (en) * | 1981-12-23 | 1987-02-10 | Baker John R | Methods of determining fructosamine levels in blood samples |
| US4876188A (en) * | 1986-11-18 | 1989-10-24 | Scripps Clinic And Research Foundation | Novel immunochemical method for assaying stable glycosylated hemoglobin |
| US5110745A (en) * | 1989-06-01 | 1992-05-05 | The Trustees Of The University Of Pennsylvania | Methods of detecting glycated proteins |
| WO1995024651A1 (en) * | 1994-03-11 | 1995-09-14 | Abbott Laboratories | Cyanide-free reagent and method for the determination of hemoglobin |
| US5484735A (en) * | 1989-08-23 | 1996-01-16 | Northwestern University | Immunoassay of glycosylated proteins employing antibody directed to reductively glycosylated N-terminal amino acids |
| US5571723A (en) * | 1991-02-07 | 1996-11-05 | Evans; Cody A. | Method of testing for diabetes that reduces the effect of interfering substances |
| US5807747A (en) * | 1989-06-13 | 1998-09-15 | Clinical Innovations Limited | Method and apparatus for determination of glycosylated protein |
| US5834315A (en) * | 1994-12-23 | 1998-11-10 | Coulter Corporation | Cyanide-free reagent and method for hemoglobin determination and leukocyte differentitation |
| US5958781A (en) * | 1994-07-14 | 1999-09-28 | Abbott Laboratories | Methods and reagents for cyanide-free determination of hemoglobin and leukocytes in whole blood |
| US20030003522A1 (en) * | 2001-06-29 | 2003-01-02 | International Business Machines Corporation | Method, system, and apparatus for measurement and recording of blood chemistry and other physiological measurements |
| US20050004006A1 (en) * | 2003-05-14 | 2005-01-06 | Jose Engelmayer | Lactoferrin in the treatment of diabetes mellitus |
| US20060094082A1 (en) * | 2004-10-26 | 2006-05-04 | Agennix Incorporated | Composition of lactoferrin related peptides and uses thereof |
| WO2020159599A1 (en) * | 2019-01-29 | 2020-08-06 | Siemens Healthcare Diagnostics Inc. | TURBIDITY NORMALIZATION ALGORITHM AND METHODS OF REDUCING INTRALIPID/LIPEMIA INTERFERENCE IN HEMOGLOBIN A1c ASSAYS |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1146769A (en) * | 1980-02-04 | 1983-05-24 | Beat E. Glatthaar | Process for the determination of diabetes |
| JPH0251091A (en) * | 1988-08-12 | 1990-02-21 | Sharp Corp | Schedule registering apparatus |
| DE4206932A1 (en) * | 1992-03-05 | 1993-09-09 | Boehringer Mannheim Gmbh | IMMUNOLOGICAL METHOD FOR DETERMINING A HAEMOGLOBIN DERIVATIVE |
| WO2001090735A1 (en) * | 2000-05-23 | 2001-11-29 | Koji Sode | Kit for assaying saccharified protein |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3918905A (en) * | 1973-07-20 | 1975-11-11 | Biolog Corp Of America | Diagnostic test for the determination of sickling hemoglobinopathies |
| US3958560A (en) * | 1974-11-25 | 1976-05-25 | Wayne Front March | Non-invasive automatic glucose sensor system |
| US3964865A (en) * | 1975-01-30 | 1976-06-22 | Sigma Chemical Company | Lyophilized human hemoglobin standard for colorimetric determination of total hemoglobin |
| US4077772A (en) * | 1977-05-31 | 1978-03-07 | Baxter Travenol Laboratories, Inc. | Method for the determination of hemoglobin in trace amounts |
| US4142858A (en) * | 1977-12-02 | 1979-03-06 | Isolab, Incorporated | Method to determine a diagnostic indicator of blood sugar condition, and, a liquid chromatographic microcolumn therefor |
-
1978
- 1978-12-26 US US05/973,368 patent/US4200435A/en not_active Expired - Lifetime
-
1979
- 1979-10-29 CA CA000338610A patent/CA1119081A/en not_active Expired
- 1979-10-30 ZA ZA00795808A patent/ZA795808B/en unknown
- 1979-11-01 GB GB7937929A patent/GB2038476B/en not_active Expired
- 1979-11-12 NL NL7908272A patent/NL7908272A/en not_active Application Discontinuation
- 1979-11-13 AU AU52754/79A patent/AU528008B2/en not_active Ceased
- 1979-12-14 DE DE2950457A patent/DE2950457B2/en not_active Ceased
- 1979-12-14 SE SE7910340A patent/SE445147B/en not_active IP Right Cessation
- 1979-12-21 IT IT28370/79A patent/IT1198318B/en active
- 1979-12-21 BE BE0/198711A patent/BE880817A/en not_active IP Right Cessation
- 1979-12-21 JP JP16581279A patent/JPS5587954A/en active Granted
- 1979-12-21 AT AT808579A patent/AT362534B/en not_active IP Right Cessation
- 1979-12-21 CH CH1142879A patent/CH641571A5/en not_active IP Right Cessation
- 1979-12-24 FR FR7931601A patent/FR2445527A1/en active Granted
- 1979-12-26 ES ES487265A patent/ES8103381A1/en not_active Expired
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3918905A (en) * | 1973-07-20 | 1975-11-11 | Biolog Corp Of America | Diagnostic test for the determination of sickling hemoglobinopathies |
| US3958560A (en) * | 1974-11-25 | 1976-05-25 | Wayne Front March | Non-invasive automatic glucose sensor system |
| US3964865A (en) * | 1975-01-30 | 1976-06-22 | Sigma Chemical Company | Lyophilized human hemoglobin standard for colorimetric determination of total hemoglobin |
| US4077772A (en) * | 1977-05-31 | 1978-03-07 | Baxter Travenol Laboratories, Inc. | Method for the determination of hemoglobin in trace amounts |
| US4142858A (en) * | 1977-12-02 | 1979-03-06 | Isolab, Incorporated | Method to determine a diagnostic indicator of blood sugar condition, and, a liquid chromatographic microcolumn therefor |
Non-Patent Citations (7)
| Title |
|---|
| H. F. Bunn et al., Science, 200, 21-27 (Apr. 1978). * |
| M. F. Perutz et al., Biochemistry, 17(17), 3640-3652 (1978). * |
| M. F. Perutz, Scientific American, 239(6), 92-125 (Dec. 1978). * |
| M. J. McDonald et al., J. Biol. Chem., 254(3), 702-707 (1979). * |
| R. G. Shulman et al., Quat. Rev. Biophysics, 8(3), 325-420 (1975). * |
| Ruth E. Benesh et al., Advances in Protein Chemistry, vol. 28, 211-237 (1974). * |
| S. J. Edelstein, Annual Review of Biochemistry, vol. 44, 209-232 (1975). * |
Cited By (33)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4297238A (en) * | 1978-11-22 | 1981-10-27 | Merck Patent Gesellschaft Mit Beschrankter Haftung | Hemolysing solution preparing hemolysed blood having a stabilized glucose content |
| WO1981000913A1 (en) * | 1979-09-24 | 1981-04-02 | J Graas | Microchromatographic device and method for rapid determination of a desired substance |
| US4260516A (en) * | 1979-12-07 | 1981-04-07 | Abbott Laboratories | Glycosylated hemoglobin standards |
| EP0030378A2 (en) * | 1979-12-07 | 1981-06-17 | Abbott Laboratories | Glycosylated hemoglobin standards |
| US4274978A (en) * | 1979-12-07 | 1981-06-23 | Abbott Laboratories | Standards for determining glycosylated hemoglobin |
| EP0030378A3 (en) * | 1979-12-07 | 1982-04-07 | Abbott Laboratories | Glycosylated hemoglobin standards |
| US4465774A (en) * | 1981-07-16 | 1984-08-14 | Sherwood Medical Company | Standard or control material for glysocylated or total hemoglobin determination |
| EP0072440A1 (en) * | 1981-07-16 | 1983-02-23 | Sherwood Medical Industries Inc. | Standard or control material for glycosylated or total hemoglobin determination |
| US4438204A (en) | 1981-10-16 | 1984-03-20 | Boehringer Mannheim Gmbh | Determination of glycosilated hemoglobin |
| US4642295A (en) * | 1981-12-23 | 1987-02-10 | Baker John R | Methods of determining fructosamine levels in blood samples |
| US4645742A (en) * | 1981-12-23 | 1987-02-24 | Baker John R | Materials for determining fructosamine levels in blood samples |
| US4629692A (en) * | 1982-12-06 | 1986-12-16 | Miles Laboratories, Inc. | Immunoassay for nonenzymatically glucosylated proteins and protein fragments an index of glycemia |
| US4876188A (en) * | 1986-11-18 | 1989-10-24 | Scripps Clinic And Research Foundation | Novel immunochemical method for assaying stable glycosylated hemoglobin |
| US5110745A (en) * | 1989-06-01 | 1992-05-05 | The Trustees Of The University Of Pennsylvania | Methods of detecting glycated proteins |
| US5807747A (en) * | 1989-06-13 | 1998-09-15 | Clinical Innovations Limited | Method and apparatus for determination of glycosylated protein |
| US5484735A (en) * | 1989-08-23 | 1996-01-16 | Northwestern University | Immunoassay of glycosylated proteins employing antibody directed to reductively glycosylated N-terminal amino acids |
| US5571723A (en) * | 1991-02-07 | 1996-11-05 | Evans; Cody A. | Method of testing for diabetes that reduces the effect of interfering substances |
| WO1995024651A1 (en) * | 1994-03-11 | 1995-09-14 | Abbott Laboratories | Cyanide-free reagent and method for the determination of hemoglobin |
| US5866428A (en) * | 1994-03-11 | 1999-02-02 | Abbott Laboratories | Cyanide-free reagent and method for the determination of hemoglobin |
| US5612223A (en) * | 1994-03-11 | 1997-03-18 | Abbott Laboratories | Cyanide-free reagent and method for the determination of hemoglobin |
| US6740527B1 (en) | 1994-07-14 | 2004-05-25 | Abbott Laboratories | Methods and reagents for cyanide-free determination of hemoglobin and leukocytes in whole blood |
| US5958781A (en) * | 1994-07-14 | 1999-09-28 | Abbott Laboratories | Methods and reagents for cyanide-free determination of hemoglobin and leukocytes in whole blood |
| US5834315A (en) * | 1994-12-23 | 1998-11-10 | Coulter Corporation | Cyanide-free reagent and method for hemoglobin determination and leukocyte differentitation |
| US6844149B2 (en) * | 2001-06-29 | 2005-01-18 | International Business Machines Corporation | Method, system, and apparatus for measurement and recording of blood chemistry and other physiological measurements |
| US20030003522A1 (en) * | 2001-06-29 | 2003-01-02 | International Business Machines Corporation | Method, system, and apparatus for measurement and recording of blood chemistry and other physiological measurements |
| US20050004006A1 (en) * | 2003-05-14 | 2005-01-06 | Jose Engelmayer | Lactoferrin in the treatment of diabetes mellitus |
| US7034126B2 (en) * | 2003-05-14 | 2006-04-25 | Agennix, Inc. | Lactoferrin in the treatment of diabetes mellitus |
| US20060094082A1 (en) * | 2004-10-26 | 2006-05-04 | Agennix Incorporated | Composition of lactoferrin related peptides and uses thereof |
| US7183381B2 (en) | 2004-10-26 | 2007-02-27 | Agennix, Inc. | Composition of lactoferrin related peptides and uses thereof |
| US20070142292A1 (en) * | 2004-10-26 | 2007-06-21 | Agennix Incorporated | Composition of lactoferrin related peptides and uses thereof |
| US7420033B2 (en) | 2004-10-26 | 2008-09-02 | Agennix, Inc. | Composition of lactoferrin related peptides and uses thereof |
| WO2020159599A1 (en) * | 2019-01-29 | 2020-08-06 | Siemens Healthcare Diagnostics Inc. | TURBIDITY NORMALIZATION ALGORITHM AND METHODS OF REDUCING INTRALIPID/LIPEMIA INTERFERENCE IN HEMOGLOBIN A1c ASSAYS |
| US11300577B1 (en) | 2019-01-29 | 2022-04-12 | Siemens Healthcare Diagnostics Inc. | Turbidity normalization algorithm and methods of reducing intralipid/lipemia interference in hemoglobin A1c assays |
Also Published As
| Publication number | Publication date |
|---|---|
| FR2445527B1 (en) | 1983-09-02 |
| JPS5587954A (en) | 1980-07-03 |
| SE7910340L (en) | 1980-06-27 |
| NL7908272A (en) | 1980-06-30 |
| ATA808579A (en) | 1980-10-15 |
| AU5275479A (en) | 1980-07-03 |
| DE2950457B2 (en) | 1981-04-02 |
| AT362534B (en) | 1981-05-25 |
| FR2445527A1 (en) | 1980-07-25 |
| IT1198318B (en) | 1988-12-21 |
| DE2950457A1 (en) | 1980-07-03 |
| CA1119081A (en) | 1982-03-02 |
| JPS6363865B2 (en) | 1988-12-08 |
| GB2038476A (en) | 1980-07-23 |
| SE445147B (en) | 1986-06-02 |
| ZA795808B (en) | 1980-10-29 |
| GB2038476B (en) | 1983-05-05 |
| CH641571A5 (en) | 1984-02-29 |
| AU528008B2 (en) | 1983-03-31 |
| IT7928370A0 (en) | 1979-12-21 |
| BE880817A (en) | 1980-06-23 |
| ES487265A0 (en) | 1981-02-16 |
| ES8103381A1 (en) | 1981-02-16 |
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